Calf rennet has been used as a milk-clotting enzyme for the production of cheese for many years. A milk-clotting activity of the calf rennet is mostly attributed to chymosin, which is an acid protease and has site-specific protease activity for milk casein with low site-nonspecific activity (digest specifically the peptide bond between phenylalanine at position 105 and methionine at position 106 in the amino acid sequence of κ-casein). Nonspecific protease activity is thought to lead to reduction in yield of the production of cheese and to generation of a bitter-taste peptide during ripening. From this reason, chymosin is an excellent milk-clotting enzyme.
However, decreases in calf slaughter and increases of cheese demand have made it difficult to supply the calf rennet. Nowadays, a milk-clotting enzyme derived from microorganisms such as Rhizomucor miehei and Rhizomucor pusillus, and a recombinant chymosin produced by introducing a calf chymosin gene into fungi or yeast are widely used as a milk-clotting enzyme.
The above-mentioned milk-clotting enzyme derived from a microorganism, as compared with the calf chymosin or recombinant chymosin, has a higher nonspecific protease activity. It is a problem that C/P ratio (ratio of milk-clotting activity to protease activity) which is important as characteristics of the milk-clotting enzyme is low. In order to solve such a drawback, in Rhizomucor pusillus, a variant gene of the milk-clotting enzyme obtained by site-directed mutagenesis with genetic engineering has been expressed and evaluated. In the variant, C/P ratio was improved to be better than a wild type by replacing glutamic acid at position 19 with alanine in the amino acid sequence of the milk-clotting enzyme (Non-patent document 1).
However, since the milk-clotting activity of the variant milk-clotting enzyme decreases by about 40 percent with the amino acid replacement, it has been difficult to put such an enzyme into a practical application. Thus, a milk-clotting enzyme derived from a microorganism in which C/P ratio is high and the milk-clotting activity is maintained or improved has been desired.
Moreover, acylation of the milk-clotting enzyme derived from microorganisms such as Rhizomucor pusillus and Rhizomucor miehei with dicarboxylic anhydride in order to improve C/P ratio has been attempted (Patent document 1). With this method, some improvement was obtained; however, those are not yet satisfactory.
[Patent document 1] Japanese Patent No. 2-18834B
[Non-patent document 1] J. Biochem. 129, 791-794, 2001